Characterization of a consensus-designed trans-cinnamic acid decarboxylase for styrene biosynthesis

Microbial biosynthesis of aromatic compounds offers significant advantages over petrochemical methods, which rely on fossil fuels and high energy inputs. Microbial fermentations occur at room temperature and ambient pressure, reducing carbon emissions and energy consumption by up to 90%. Genetic engineering of microbial chassis is key to optimizing biosynthetic processes, enabling efficient production of aromatic compounds from sugars. However, the intrinsic toxicity of these compounds presents challenges. Pseudomonas putida DOT-T1E, known for its tolerance to solvents, is ideal for producing toxic compounds. Styrene biosynthesis involves converting phenylalanine into trans-cinnamate via phenylalanine ammonia lyase enzymes, followed by decarboxylation to styrene. This second step is challenging, as trans-cinnamate decarboxylases have only been described in fungi. PSC1, a consensus protein designed from multiple alignments of fungal ferulate decarboxylases, enables styrene production in Pseudomonas. PSC1 is a globular dimer with a molecular mass of 104.7 kDa, high thermal stability (T_m 63°C), and activity at temperatures up to 50°C. The crystal structure of PSC1, determined at 2.1 Å, reveals a homodimer with three domains per monomer. A hydrophobic pocket in domain 2, essential for cofactor and substrate binding, was identified. Mutagenesis shows that Arg175, Glu280, and Glu285 are critical for catalysis, as replacing them with alanine abolished the decarboxylation.IMPORTANCEThe petrochemical industry is highly polluting due to its use of extremely high temperatures, high pressure, and toxic catalysts. Synthetic biology offers an alternative by enabling the production of many chemicals through cell factories that operate at room temperature and ambient pressure, potentially reducing CO₂ emissions by up to 90%. We have engineered a solvent-tolerant Pseudomonas strain to produce styrene from L-phenylalanine in a two-step process. For the second step, we designed a de novo consensus protein that operates efficiently. In this study, we present its physico-chemical properties and unveil its 3D structure.